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Abstract

Background

Parkinson’s disease (PD) is a devastating neurodegenerative disorder characterized
by progressive motor debilitation, which affects several million people worldwide.
Recent evidence suggests that glial cell activation and its inflammatory response
may contribute to the progressive degeneration of dopaminergic neurons in PD. Currently,
there are no neuroprotective agents available that can effectively slow the disease
progression. Herein, we evaluated the anti-inflammatory and antioxidant efficacy of
diapocynin, an oxidative metabolite of the naturally occurring agent apocynin, in
a pre-clinical 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of
PD.

Methods

Both pre-treatment and post-treatment of diapocynin were tested in the MPTP mouse
model of PD. Diapocynin was administered via oral gavage to MPTP-treated mice. Following
the treatment, behavioral, neurochemical and immunohistological studies were performed.
Neuroinflammatory markers, such as ionized calcium binding adaptor molecule 1 (Iba-1),
glial fibrillary acidic protein (GFAP), gp91phox and inducible nitric oxide synthase
(iNOS), were measured in the nigrostriatal system. Nigral tyrosine hydroxylase (TH)-positive
neurons as well as oxidative markers 3-nitrotyrosine (3-NT), 4-hydroxynonenal (4-HNE)
and striatal dopamine levels were quantified for assessment of the neuroprotective
efficacy of diapocynin.

Results

Oral administration of diapocynin significantly attenuated MPTP-induced microglial
and astroglial cell activation in the substantia nigra (SN). MPTP-induced expression
of gp91phox and iNOS activation in the glial cells of SN was also completely blocked
by diapocynin. Notably, diapocynin markedly inhibited MPTP-induced oxidative markers
including 3-NT and 4-HNE levels in the SN. Treatment with diapocynin also significantly
improved locomotor activity, restored dopamine and its metabolites, and protected
dopaminergic neurons and their nerve terminals in this pre-clinical model of PD. Importantly,
diapocynin administered 3 days after initiation of the disease restored the neurochemical
deficits. Diapocynin also halted the disease progression in a chronic mouse model
of PD.

Conclusions

Collectively, these results demonstrate that diapocynin exhibits profound neuroprotective
effects in a pre-clinical animal model of PD by attenuating oxidative damage and neuroinflammatory
responses. These findings may have important translational implications for treating
PD patients.

Keywords:

Background

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, estimated
to affect 1% of the population over 65 years of age. It is a chronic and progressive
disease characterized predominantly by resting tremors, bradykinesia, muscular rigidity
and postural instability, along with several non-motor symptoms
[1]. The pathological hallmarks of PD are the depletion of striatal dopamine caused by
degeneration of dopaminergic neurons in the substantia nigra (SN) region of the midbrain,
appearance of cytoplasmic inclusions, known as Lewy bodies in surviving neurons of
the SN, and activation of glial cells
[2,3].

Although the etiologic mechanisms of PD are poorly understood, recent reports implicate
brain inflammation and oxidative stress play an important role in disease pathogenesis
[2,4]. Microglia and astrocytes are major mediators of neuroinflammation in PD. Several
reports have demonstrated the activation of microglial cells and astroglial cells
in close proximity to the damaged or dying dopaminergic neurons in SN
[5]. Levels of nitrite (NO2−), a metabolite of nitric oxide (·NO and inducible nitric oxide synthase (iNOS) are higher in the central nervous system
of human PD cases and in animal models of PD
[6]. Consistent with this finding, iNOS knockout animals were resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-induced dopaminergic neuronal loss in the SN
[7].

One of the major sources of reactive oxygen species (ROS) in neurodegeneration is
NADPH oxidase, a multimeric enzyme that generates both superoxide O2− (O2 and H2O2[8]. The reaction between O2− and ·NO forms peroxynitrite (ONOO-), another key player of dopaminergic neurodegeneration in PD. Moreover, 4-hydroxynonenal
(4-HNE), an unsaturated aldehyde derived from lipid hydroperoxidase, is reported to
mediate the induction of neuronal apoptosis in the presence of oxidative stress
[9]. Collectively, these findings strongly suggest that mitigation of neuroinflammation
and oxidative stress may be a viable neuroprotective strategy for treatment of PD.

Several inhibitors of NADPH oxidase have been tested for their anti-inflammatory and
antioxidant effects in dopaminergic cells
[10]. For example, apocynin (4-hydroxy-3-methoxyacetophenone), a plant-derived antioxidant,
has been widely used as an NADPH oxidase inhibitor in in vitro and in vivo experimental models of PD
[10-12]. However, apocynin failed to protect dopaminergic neurons against rotenone-mediated
neurotoxicity in the absence of glial cells
[4]. In vivo, apocynin has been shown to form diapocynin, a dimer of apocynin, resulting in the
inhibition of NADPH oxidase
[13].

Thus, in the present study, we synthesized the dimeric derivative diapocynin and tested
its antioxidant and anti-inflammatory efficacies in mouse models of PD. The results
presented here show that diapocynin suppresses MPTP-induced glial activation, attenuates
nigral expression of proinflammatory molecules, reduces oxidative stress and protects
the nigrostriatal axis after MPTP administration. Collectively, these results suggest
that additional pre-clinical development of diapocynin may yield an effective neuroprotective
and anti-neuroinflammatory drug capable of arresting the progression of PD.

Methods

Animals and treatment

Eight-week-old male C57BL/6 mice weighing 24 to 28 g were housed in standard conditions:
constant temperature (22 ± 1°C), humidity (relative, 30%) and a 12 h light/dark cycle.
Use of the animals and protocol procedures were approved and supervised by the Institutional
Animal Care and Use Committee (IACUC) at Iowa State University (Ames, IA, USA) To
assess the neuroprotective effect of diapocynin, we first used low doses of diapocynin
(100 and 150 mg/kg/day) via oral gavage, but these doses showed only a moderate effect
in attenuating MPTP-induced neurochemical deficits. Therefore, we used a 300 mg/kg
dose of diapocynin in the subacute MPTP model of PD for detailed characterization
of the neuroprotective efficacy of diapocynin. This 300 mg/kg dose was chosen based
on apocynin, which has been used at a similar dose range in amyotrophic lateral sclerosis
(ALS) and Alzheimer's disease mouse models
[14].

In the subacute MPTP regimen, mice received 25 mg/kg/day MPTP-HCl in saline intraperitoneally
for 5 consecutive days. Diapocynin was dissolved in 10% ethanol 1 day before the MPTP
insult and the drug treatment continued for 11 days. Animals were subjected to measurements
of inflammatory markers, neurotransmitter levels, behavioral changes and neuronal
damage at various time points. Control mice received equivolumes of the vehicle solution.

In the post-treatment regimen, diapocynin was administered 3 days after the MPTP treatment.
For chronic MPTP treatment, mice received 2 doses of MPTP (25 mg/kg/dose, s.c.) and
2 doses of probenecid (250 mg/kg/dose, i.p.) per week for 5 consecutive weeks. Mice
received 3 doses of diapocynin (100 mg/kg/day) per week by oral gavage, and the drug
treatment started 1 week before MPTP injections, continued throughout the MPTP injection
period and extended for another 45 days of post-MPTP treatment. After all treatments,
animals were subjected to behavioral, neurochemical and histological measurements.

Samples were prepared and quantified, as described previously
[15,16]. On the day of analysis, neurotransmitters from striatal tissues were extracted using
an antioxidant extraction solution (0.1 M perchloric acid, containing 0.05% Na2EDTA and 0.1% Na2S2O5) and isoproterenol (as internal standard). Dopamine, 3,4-dihydroxyphenyl-acetic acid
(DOPAC) and homovanillic acid (HVA) were separated isocratically by a reversed-phase
column with a flow rate of 0.6 ml/min. An HPLC system (ESA, Inc, Bedford, MA, USA)
with an automatic sampler equipped with refrigerated temperature control (Model 542;
ESA, Inc) was used for these experiments.

HPLC/mass spectrometry (MS) analysis of diapocynin

Diapocynin from the striatum and SN was quantified using the Agilent 1100 Series LC/MS
binary pump (Agilent, Santa Clara, CA, USA), PDA detector (UV diode array detector)
and an autosampler. On the day of analysis, a 20 μl sample was passed through the
0.2 μm filter at the eluent flow rate of 0.25 ml/min. Negative-ion, atmospheric pressure
chemical ionization was used at amplitude 1.5 volts, and manual MS/MS was done. The
mobile phase used in LC/MS consisted of a gradient elution. Solvent A was 480:20:0.38
water:methanol:ammonium acetate (v/v/w) and solvent B was 20:480:0.38 water:methanol:ammonium
acetate (v/v/w). The standards series were taken as 0.3 μg, 1.0 μg, 3.0 μg, 10 μg,
and 30 μg. The actual molecular weight of diapocynin is 329.1 g/mol, but by breaking
the molecule in MS/MS it becomes 313.9 g/mol by elimination of one methyl molecule.
The retention time for the diapocynin peak was 1.9 minutes. Data were fit to a straight
line by linear regression analysis using Quant analysis software (Agilent, Santa Clara,
CA, USA).

Fluoro-Jade B (FJB) and TH double labeling

On the day of staining, sections were incubated with anti-TH antibody (Chemicon),
followed by Alexa Fluor 568 donkey anti-mouse (Invitrogen) secondary antibody. Then
FJB staining was done on the same sections by the modified FJB stain protocol, including
incubation in 0.06% potassium permanganate for 2 minutes and 0.0002% FJB stain for
5 minutes. Sections were viewed under a Nikon inverted fluorescence microscope (Model
TE-2000U; Nikon) and images were captured with a SPOT digital camera (Diagnostic Instruments,
Inc).

Behavioral measurements

An automated device (Model RXYZCM-16; Accuscan, Columbus, OH, USA) was used to measure
the spontaneous activity of mice. The activity chamber was 40 × 40 × 30.5 cm, made
of clear Plexiglas and covered with a Plexiglas lid with holes for ventilation. Data
were collected and analyzed by a VersaMax Analyzer (Model CDA-8; AccuScan). Before
any treatment, mice were placed inside the infrared monitor for 10 minutes daily for
3 consecutive days to train them. Five days after the last MPTP injection, open field
and rotarod experiments were conducted. Locomotor activities were recorded for 10
minute test sessions. A speed of 20 rpm was used in the rotarod experiment. Mice were
given a 7 to 10 minute rest interval to prevent stress and fatigue.

Data analysis

Data analysis was performed using Prism 4.0 software (GraphPad Software, Inc, San
Diego, CA, USA). Raw data were first analyzed using one-way analysis of variance and
then Tukey’s post-test was performed to compare all treatment groups. Differences
with P <0.05 were considered significant.

Results

Male C57BL/6 mice were treated with diapocynin by oral gavage daily and then inflammatory,
neurochemical, behavioral and histological studies were performed at various time
intervals, as depicted in Figure
1A. To assess the neuroprotective effect of diapocynin, we first used two lower doses
of diapocynin (100 and 150 mg/kg/day) and measured the dopamine level from striatum
(Figure
1B). Both 100 and 150 mg/kg/day doses of diapocynin afforded some protection against
MPTP-induced striatal dopamine loss, but it was not significant (Figure
1B). Therefore, we increased the dosage of diapocynin to 300 mg/kg/day and then evaluated
the neuroprotective effects. Before determining the neuroprotective properties of
diapocynin, the ability of diapocynin to cross the blood-brain barrier was examined.
The LC/MS results showed a significant accumulation of diapocynin in the SN and striatum
of the brain following the oral gavage treatment of diapocynin (300 mg/kg/day) (Figure
1C,D).

Figure 1.Diapocynin enters brain and attenuates MPTP-induced depletion of neurotransmitters. (A) Treatment schedule of MPTP-injected mice with diapocynin. (B) Mice were treated with two doses of diapocynin (100 and 150 mg/kg/day) and seven
days after the last dose of MPTP treatment, striatal dopamine level was measured using
high-performance liquid chromatography (HPLC). Quantification of diapocynin in the
substantia nigra (SN) and striatum of mice. Mice were administered diapocynin (300
mg/kg/day) by oral gavage 24 h before MPTP treatment, and co-treatment with MPTP (25
mg/kg/day) was continued for 5 days, and post-treatment with MPTP (25 mg/kg/day) lasted
6 days. Seven days after the last injection of MPTP, SN and striatum were dissected
out and quantified for diapocynin. (C) Standard curve of diapocynin standards ranging from 0.3 μg to 30 μg. (D) Quantification of diapocynin in SN and striatum. Seven days after the last MPTP
treatment, striatal (E) dopamine, (F) 3,4-dihydroxyphenyl-acetic acid (DOPAC) and (G) homovanillic acid (HVA) were measured by HPLC. Data are means ± SEM of eight to
ten mice per group. ***P <0.001 versus the control group; *P <0.05 vs the MPTP group; #P <0.01 vs the MPTP group; aP <0.01 vs the control group.

Following 300 mg/kg/day diapocynin treatment, mice were sacrificed and brains were
processed for HPLC neurochemical analysis (Figure
1E,G). We quantified levels of dopamine and its metabolites, DOPAC and HVA, in striatum
following diapocynin treatment. As shown in Figure
1E, MPTP injection led to a >75% reduction in striatal dopamine levels compared with
the striata of control mice. Remarkably, diapocynin treatment significantly protected
against MPTP-induced striatal dopamine depletion (Figure
1E). Diapocynin treatment also significantly restored DOPAC and HVA levels in MPTP-treated
mice (Figure
1F,G).

In order to determine whether diapocynin interferes with the toxic metabolic conversion
of MPTP to MPP+ by MAO-B, we measured the level of MPP+ in striatum 3 h after the final MPTP injection. We found that diapocynin had no effect
on striatal levels of MPP+ (MPTP mice, 1860 ± 569 ng/g; MPTP plus diapocynin mice, 1775 ± 586 ng/g). Next, we
also tested whether diapocynin treatment alone alters neurochemical levels in the
striatum. As shown in Figure
1E,G, oral administration of diapocynin (300 mg/kg/day) alone for 12 days did not alter
striatal dopamine levels. Also, diapocynin treatment did not produce behavioral abnormalities
(data not shown).

Diapocynin inhibits MPTP-induced glial activation in the SN

Next, we determined if diapocynin protected against MPTP-induced microglial activation
and astrogliosis. As shown in Figure
2, a significant increase in the expression of ionized calcium binding adaptor molecule
1 (Iba-1) and glial fibrillary acidic protein (GFAP) in SN of the MPTP-treated group
was observed. DAB immunostaining of Iba-1 in MPTP nigral brain tissue showed an increased
number of amoeboid-shaped microglial cells with thick processes, indicative of active
microgliosis (Figure
2A). DAB immunostaining of GFAP in MPTP-treated mice showed excessive astrogliosis,
as measured by increased size and processes thickness in GFAP-positive cells (Figure
2D).

Importantly, diapocynin strongly inhibited MPTP-induced microgliosis, as demonstrated
by the very few Iba-1-positive microglial cells in the drug-treated group (Figure
2A). Also, diapocynin significantly decreased MPTP-induced increases in GFAP-positive
astroglial cells in the mouse SN (Figure
2D). Western blot analysis for Iba-1 and GFAP also revealed that diapocynin suppresses
nigral Iba-1 (Figure
2B and C) and GFAP (Figure
2E,F) protein expression in MPTP-treated mice. Together, these data suggest that diapocynin
significantly attenuates glial cell activation in the nigral regions of the MPTP mouse
model of PD.

Diapocynin inhibits MPTP-induced iNOS expression in mouse SN

iNOS, a key proinflammatory enzyme, is typically elevated in disease conditions
[3]. We observed a marked increase in the expression of iNOS in the SN of MPTP-treated
mice as compared to control mice (Figure
3A,B). However, diapocynin attenuated MPTP-induced expression of iNOS protein (Figure
3A,B). Additionally, immunofluorescence analysis for iNOS in the SN sections shows
that MPTP treatment led to a marked increase in nigral iNOS protein expression, and
that iNOS co-localized with GFAP-positive astrocytes and Iba-1-positive microglial
cells (Figure
3C,D). Consistent with its inhibitory effect on glial cell activation, diapocynin suppressed
MPTP-induced expression of iNOS (Figure
3C,D). These results demonstrate that diapocynin effectively suppresses the expression
of the proinflammatory molecule iNOS in vivo in the SN of MPTP-treated mice.

Diapocynin inhibits formation of nitrotyrosine and hydroxynonenal in the nigral dopaminergic
neurons of MPTP-treated mice

3-Nitrotyrosine (3-NT) has been widely used as a marker of nitric oxide-dependent
oxidative stress
[21]. Western blot analysis demonstrated increased expression of 3-NT modified proteins
in the SN of MPTP-treated mice, while diapocynin treatment significantly suppressed
MPTP-induced 3-NT levels (Figure
5A,B). Further confirmation came from immunolabeling of 3-NT in the SN sections. In
sections from MPTP-treated mice, a dramatic increase in the expression of 3-NT, specifically
in the SN region of the ventral midbrain, was observed. Notably, 3-NT co-localized
(yellow color) with TH-positive dopaminergic neurons (Figure
5C). However, 3-NT expression was observed in very few TH-positive dopaminergic neurons
in the MPTP and diapocynin co-treated animals (Figure
5C). These results strongly suggest that diapocynin inhibits nitration of TH-positive
dopaminergic neurons in the nigra during neurotoxic insult.

Following characterization of the neuroprotective effect of diapocynin in the typical
subacute MPTP model, we further examined whether diapocynin can intervene the on-going
degenerative processes in a post-treatment paradigm. In order to test the efficacy
of diapocynin post-treatment, mice were treated with MPTP at a dose of 25 mg/kg/day
for 3 days followed by co-treatment with MPTP and diapocynin (300 mg/kg/day) for 2
days. Mice also received another 6 doses of diapocynin (300 mg/kg/day) and were sacrificed
for neurotransmitter analysis. As evident from Figure
8A,B,C, we observed significantly reduced striatal dopamine (75%), DOPAC (73%) and
HVA (70%) in MPTP-treated mice. Importantly, diapocynin post-treatment showed a reduction
of 52% of dopamine (Figure
8A), 50% of DOPAC (Figure
8B) and 40% of HVA (Figure
8C). Thus, these results suggest that diapocynin slows the progression of dopaminergic
neurodegeneration in a post-treatment MPTP mouse model of PD.

Figure 8.Diapocynin suppresses disease progression in the subacute MPTP mouse model of Parkinson’s
disease (PD) and protects striatal neurotransmitter depletion in the chronic MPTP
mouse model of PD. For disease progression study, mice were treated with MPTP (25 mg/kg/day) for 5 days.
Diapocynin (300 mg/kg/day) treatment started on the 4th day of MPTP injection and
continued for another 8 days. Mice were sacrificed 1 day after the last dose of diapocynin
and striatal (A) dopamine, (B) 3,4-dihydroxyphenyl-acetic acid (DOPAC) and (C) homovanillic acid (HVA) were measured by high-performance liquid chromatography
(HPLC). For chronic treatment, mice were treated with 2 doses of MPTP (25 mg/kg/day,
s.c.) and 2 doses of probenecid (250 mg/kg/day, i.p.) per week for 5 weeks. Control
mice received only saline. One week prior to MPTP/probenecid treatment, one group
of mice received 3 doses of diapocynin (100 mg/kg/day, gavage) and this treatment
continued for 12 consecutive weeks. Another batch of mice received 3 doses of diapocynin
(100 mg/kg/day, gavage) in a week for consecutive 12 weeks. Immediately after treatment,
mice were sacrificed and striatal (D) dopamine, (E) DOPAC and (F) HVA were measured by HPLC. Data are means ± SEM of 8 to 10 mice per group. ***P <0.001 vs the control group; **P <0.01 vs the MPTP group; *P <0.05 vs the MPTP group.

Discussion

Neuroinflammation and oxidative stress are now well recognized as key pathophysiological
events contributing to the progressive loss of nigral dopaminergic neurons in PD
[2-4]. However, an effective neuroprotective therapy to halt the progression of the disease
is not available. Here, we report the anti-inflammatory and antioxidative properties
of a synthetic analog of apocynin in the MPTP mouse model of PD. Recent studies have
shown conversion of apocynin to diapocynin (apocynin dimer) in vivo, which prevents the assembly and activation of the NADPH oxidase complex
[13]. Additionally, diapocynin is 13-fold more lipophilic than apocynin
[22]. Here, we show that diapocynin inhibits MPTP-induced activation and expression of
both iNOS and gp91phox in activated glial cells, suggesting that diapocynin has anti-inflammatory
properties against neurotoxic stress. Diapocynin also attenuates the formation of
ONOO- and 4-HNE in dopaminergic neurons in response to various stimuli, further confirming
the antioxidant properties of this compound.

Importantly, diapocynin also protects against MPTP-induced motor deficits, striatal
neurotransmitter depletion and nigrostriatal degeneration. Furthermore, diapocynin
is effective in post-treatment paradigms as well as in chronic neurodegenerative models
of PD. Derivatives of natural compounds, such as diapocynin, are a key translational
approach for the development of therapies for PD. To our knowledge, this is the first
report showing anti-inflammatory, antioxidative and neuroprotective properties of
a novel apocynin derivative in animal models of PD.

NADPH oxidase has emerged as a major source of oxidative stress in the brain, particularly
in neurodegenerative disorders, such as PD, Alzheimer's disease, ALS and multiple
sclerosis
[23]. Apocynin has been shown to inhibit NADPH oxidase, which generates ROS during inflammatory
processes
[10]. Although the mechanism of inhibition of apocynin is not clear, it is thought to
prevent the recruitment of cytosolic NADPH oxidase subunit p47phox to the membrane,
thereby inhibiting NADPH oxidase activity. Apocynin has been shown to attenuate superoxide
formation and oxidative stress in vivo as well as reduce acute inflammation in lung and spinal cord
[24-26]. Furthermore, apocynin administered at a dose of 300 mg/kg/day protects against oxidative
damage induced by cerebral ischemia
[27] and ALS
[14].

In contrast, recent studies have shown that apocynin failed to show any improvement
in transgenic animal models of Alzheimer's disease
[28] or ALS
[29]. In vitro studies in dopaminergic neuronal cell lines and primary cultures also demonstrated
a protective role of apocynin in 1-methyl-4-phenyl-pyridinium ion (MPP+) or MPTP-induced NADPH oxidase mediated apoptotic cell death
[10,30]. Also, a pro-oxidative nature of apocynin has been shown in non-phagocytic cells,
where it increases ROS production significantly
[31]. Thus, these studies suggest that the development of an improved apocynin related
compound may yield a better neuroprotective agent for treatment of PD.

In the central nervous system, glial activation involving astrocytes, microglial cells,
lymphocyte infiltration, and production of proinflammatory mediators including cytokines,
chemokines, prostaglandins, and reactive mediators, such as reactive nitrogen species
(RNS) and ROS, are all hallmarks of inflammatory reactions. MPP+, the active metabolite of MPTP, is believed to be responsible for glial activation
mediated inflammation and neurodegeneration
[2]. In our study, we also observed marked activation of microglia and astrocytes, measured
by Western blotting and immunohistochemistry after MPTP treatment in SN, and diapocynin
significantly attenuated MPTP-induced microgliosis and astrogliosis (Figure
2).

Nuclear factor kappa B (NF-κB), a transcription factor, has been shown to be an important
regulator of the microglial and astroglial proinflammatory reactions in the SN. The
promoter regions of proinflammatory molecules, including iNOS, contain the binding
sites for NF-ĸB
[19]. Astroglia and microglia in the healthy brain do not express iNOS, but following
toxic or inflammatory damage, reactive astroglia and microglia express iNOS in the
brain
[32]. Studies have shown that MPTP treatment produces significantly reduced neuronal loss
in mice deficient in iNOS compared to their wild-type counterparts
[33].

In this study, we demonstrate that diapocynin, a pharmacological inhibitor of microglial
NADPH oxidase, effectively attenuates MPTP-induced increases in iNOS expression (Figure
3), suggesting the potential use of diapocynin as an anti-inflammatory agent. RNS as
well as ROS play a pivotal role in oxidative stress and inflammation in PD. NADPH
oxidase, which is a major ROS-producing enzyme of microglial cells, mediates superoxide
production and controls the levels of pro-inflammatory neurotoxic factors, such as
TNFα and IL-1β
[34]. In our study, we demonstrate that diapocynin attenuates MPTP-induced expression
of microglial gp91phox in SN and thereby reduces the production of ROS (Figure
4).

Besides having direct toxic effects on nigral dopaminergic neurons, nitric oxide (·NO) and superoxide O2− derived from astrocytes and microglia can react to form the highly reactive nitrogen
species peroxynitrite (ONOO-). Peroxynitrite causes nitration of tyrosine residues in various proteins including
TH and α-synuclein
[21,35]. Peroxinitrite mediated nitration of TH is associated with reduced enzymatic activity.

3-NT is widely used as a marker of nitrative damage. Here, we found increased expression
of 3-NT in dopaminergic neurons in SN of MPTP-treated mice, predominantly co-localized
with TH-positive dopaminergic neurons (Figure
5A,B,C). However, diapocynin significantly decreased the MPTP-induced increase in 3-NT
in dopaminergic neurons in the SN.

Along with peroxynitrite, levels of 4-HNE, an unsaturated aldehyde generated during
lipid peroxidation, were also significantly increased in the SN of PD brains compared
to controls
[36]. 4-HNE has been demonstrated to block mitochondrial respiration and induce caspase-dependent
apoptosis
[37,38]. In our study, we showed increased expression of 4-HNE, a marker of oxidative damage
in the SN of MPTP-treated mice, which was colocalized in the cytosol of TH-positive
dopaminergic neurons (Figure
5D,E,F). However, diapocynin significantly decreased the amount of 4-HNE in the MPTP-treated
SN, indicating that diapocynin acted by attenuating ROS generation.

The lack of an effective therapy to halt the progression of PD has been a longstanding
challenge in the field. Administration of a dopamine agonist or levodopa has been
the leading treatment for PD symptoms, but these treatments do not affect disease
progression. Various putative neuroprotective agents, including glial cell line-derived
neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), TGF-β and other
small molecule compounds, have been tested in animal models of PD
[39,40]. However, most of these compounds failed in either pre-clinical trials or human trials
due to their inability to cross the blood-brain barrier or due to limited bioavailability.
Moreover, they also caused adverse side effects. Hence, understanding the mechanism
of the disease process and development of a successful neuroprotective therapeutic
approach to halt the disease progression are of principal importance in PD research.

Diapocynin has several advantages compared to other experimental drugs, including
its parent compound apocynin. These include: (1) co-treatment of diapocynin and MPTP
profoundly attenuated MPTP-induced glial activation and proinflammatory events, (2)
diapocynin suppressed oxidative stress in vivo in the SN of MPTP-treated mice, (3) diapocynin treatment improved MPTP-induced behavioral
deficits, (4) diapocynin protected TH-positive dopaminergic neurons from MPTP toxicity
and restored the level of dopamine and its metabolites, and (5) oral administration
of diapocynin on day 4, after the disease has been initiated by MPTP, also restored
the levels of striatal dopamine neurotransmitters in MPTP-treated mice, suggesting
that diapocynin could attenuate disease progression.

It is noteworthy that diapocynin does not interfere with MPTP metabolism, demonstrating
the true neuroprotective effect in the MPTP model. Also, diapocynin is fairly nontoxic,
as the mice treated with diapocynin alone (300 mg/kg/day) for 12 days did not show
any sign of behavioral imparities and their neurotransmitter levels were not different
from the saline-treated control mice (Figures
1E,F,G,H and
8D,E,F). Another advantage is that diapocynin can be administered orally by gavage.
Being a lipophilic molecule, diapocynin easily crosses the blood-brain barrier and
enters the SN (>1.5 μg/mg tissue) and striatum (>0.9 μg/mg tissue) regions of brain,
as detected by LC/MS/MS (Figure
1C and D). We had to use 300 mg/kg oral dose in order to achieve a desired neuroprotective
effect. Although the exact reason for the requirement of a high dose of diapocynin
is not clear, it is possible that diapocynin rapidly undergoes metabolic degradation
similar to apocynin
[41]. Nevertheless, future studies are needed to clarify the metabolic fate of diapocynin
in vivo. Taken together, our results demonstrate that diapocynin is a promising neuroprotective
agent that deserves further exploration for its use in clinical settings.

Conclusions

In summary, our results demonstrate that oral administration of diapocynin, a metabolite
of apocynin, attenuates key neuroinflammatory events, including microglial and astroglial
activation, iNOS upregulation, and oxidative and nitrative damage, in a MPTP mouse
model of PD. Importantly, diapocynin treatment protects against nigral dopaminergic
neuronal damage and behavioral deficits in the animal model of PD. This systematic
characterization of the anti-inflammatory and neuroprotective efficacy of diapocynin
in pre-clinical models of PD will facilitate further exploration of the compound for
clinical application in the future.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AG and AGK designed research. AG, JJ and PS performed research. AG and AGK analyzed
data. AG, BK, AGK wrote the paper. AG, AK, VA, BPD, BK and AGK were involved in editing
drafts of the manuscript. All authors approved the final manuscript.

Acknowledgements

The authors would like to thank Alison Gifford of the Medical College of Wisconsin
(Milwaukee, WI, USA), for her help in the study. This study was supported by grants
from the National Institutes of Health NS039958 (BK, AGK), NS65167 (AK) and the Parkinson’s
Foundation (BK). W Eugene and Linda Lloyd Endowed Chair to AGK, and Harry R and Angeline
E Quadracci Professor in Parkinson’s Research to BK, are also acknowledged.